U.S. patent application number 16/090637 was filed with the patent office on 2019-04-18 for water-soluble iron ion fluorescent probe and preparation method thereof.
The applicant listed for this patent is CHANGZHOU VOCATIONAL INSTITUTE OF ENGINEERING. Invention is credited to Bai CHEN, Wenhua CHEN, Yaozhong LI, Qiaoyun LIU, Wenbin LIU, Xiuxiu QI.
Application Number | 20190112483 16/090637 |
Document ID | / |
Family ID | 60785856 |
Filed Date | 2019-04-18 |
United States Patent
Application |
20190112483 |
Kind Code |
A1 |
QI; Xiuxiu ; et al. |
April 18, 2019 |
WATER-SOLUBLE IRON ION FLUORESCENT PROBE AND PREPARATION METHOD
THEREOF
Abstract
The present invention relates to a water-soluble ferric ion
fluorescent probe, and the fluorescent probe molecule has a
structure as shown in the following figure. The present invention
also discloses a preparation method of the water-soluble ferric ion
fluorescent probe. ##STR00001##
Inventors: |
QI; Xiuxiu; (Changzhou,
Jiangsu, CN) ; CHEN; Wenhua; (Changzhou, Jiangsu,
CN) ; CHEN; Bai; (Changzhou, Jiangsu, CN) ;
LIU; Qiaoyun; (Changzhou, Jiangsu, CN) ; LI;
Yaozhong; (Changzhou, Jiangsu, CN) ; LIU; Wenbin;
(Changzhou, Jiangsu, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHANGZHOU VOCATIONAL INSTITUTE OF ENGINEERING |
Changzhou, Jiangsu |
|
CN |
|
|
Family ID: |
60785856 |
Appl. No.: |
16/090637 |
Filed: |
June 30, 2016 |
PCT Filed: |
June 30, 2016 |
PCT NO: |
PCT/CN2016/087965 |
371 Date: |
October 2, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09B 11/24 20130101;
C09K 11/06 20130101; C07D 491/107 20130101; C09B 69/103 20130101;
G01N 33/182 20130101; G01N 21/643 20130101 |
International
Class: |
C09B 11/24 20060101
C09B011/24; G01N 21/64 20060101 G01N021/64; G01N 33/18 20060101
G01N033/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2016 |
CN |
201610497836.6 |
Claims
1. A water-soluble ferric ion fluorescent probe, characterized by
having the following structure: ##STR00004##
2. A preparation method of the water-soluble ferric ion fluorescent
probe according to claim 1, comprising the following steps: (1)
using ethanol as a solvent, adding rhodamine 6G to a concentration
of 0.02 mol/L to 0.2 mol/L, adding 1,3-propanediamine and reacting
therewith, wherein the molar ratio of 1,3-propanediamine to
rhodamine 6G is 0.5:1 to 10:1, controlling the reaction temperature
at 40 to 80.degree. C., and reacting for 4-9 h, cooling to
precipitate a precipitate, and recrystallizing with acetonitrile to
obtain a white crystal A; (2) using dichloromethane as a solvent,
adding methoxy polyethylene glycol MPEG2000 to a concentration of
0.025 mol/L to 0.25 mol/L, adding triethylamine in a molar ratio of
triethylamine to methoxy polyethylene glycol MPEG2000 of 10:1 to
1:1, dropwise adding chloroacetyl chloride in a molar ratio of
chloroacetyl chloride to methoxy polyethylene glycol MPEG2000 of
10:1 to 1:1, stirring in the dark overnight, precipitating in
diethyl ether to obtain a product, dissolving the product in water,
adjusting the pH value to 6, extracting 3 times with 20 ml of
dichloromethane, and adding diethyl ether and washing to obtain a
precipitate B; and (3) using dichloromethane as a solvent, adding
the white crystal A and the precipitate B in a molar ratio of 0.2:1
to 1:0.2, and adding KI in a molar ratio of KI to the white crystal
A of 0.1:1 to 1:0.1, and adding potassium carbonate of 0.002 mol/L
to 0.2 mol/L, reacting for 2-48 h, filtering, and adding filtrate
to diethyl ether to precipitate a precipitate C which is the
water-soluble ferric ion fluorescent probe.
Description
TECHNICAL FIELD
[0001] The present invention belongs to the field of new materials
for ion probes and particularly relates to an OFF-ON type ferric
ion fluorescent probe and a preparation method thereof, and the
probe can be used for the determination of ferric ions in an
all-water system.
BACKGROUND ART
[0002] Ferric iron is one of the necessary ions in living
organisms, which plays a role in transporting and storing oxygen in
the body and has extremely important physiological significance. On
the other hand, although iron is an essential element of the body,
excessive iron in the human body produces excessive oxygen
radicals, which may cause damage to the body. Therefore, it is
necessary to develop a Fe.sup.3+ fluorescent probe with excellent
performance in order to dynamically detect the content and
distribution of Fe.sup.3+ in a living organism or environment in
real time and online. At present, most of the ferric ion
fluorescent probes are poorly water-soluble, and a certain
proportion of organic solvents need to be added, which greatly
limits their application, and particularly limits the use in an
aqueous environment containing living organisms because of the lack
of such organic solvents in the living organisms. If an organic
solvent is additionally added, it may cause damage to living
organisms because organic solvents are often toxic.
[0003] Therefore, there is an urgent need to develop a
water-soluble ferric ion fluorescent probe that can be used in an
all-water environment.
SUMMARY OF THE INVENTION
[0004] An object of the present invention is to provide a synthetic
method of a novel OFF-ON type rhodamine derivative water-soluble
ferric ion fluorescent probe. This method gives a soluble rhodamine
derivative which has a good application prospect in the detection
of ferric ion in an all-water environment.
[0005] A first aspect of the present invention relates to a
water-soluble ferric ion fluorescent probe, and the fluorescent
probe molecule has a structure as shown in the following
figure:
##STR00002##
[0006] A second aspect of the present invention relates to a
preparation method of the water-soluble ferric ion fluorescent
probe, including the following steps:
[0007] (1) using ethanol as a solvent, adding rhodamine 6G to a
concentration of 0.02 mol/L to 0.2 mol/L, adding 1,3-propanediamine
and reacting therewith, wherein the molar ratio of
1,3-propanediamine to rhodamine 6G is 0.5:1 to 10:1, controlling
the reaction temperature at 40 to 80.degree. C., and reacting for
4-9 h. cooling to precipitate a precipitate, and recrystallizing
with acetonitrile to obtain a white crystal A;
[0008] (2) using dichloromethane as a solvent, adding methoxy
polyethylene glycol MPEG2000 to a concentration of 0.025 mol/L to
0.25 mol/L, adding triethylamine in a molar ratio of triethylamine
to methoxy polyethylene glycol MPEG2000 of 10:1 to 1:1, dropwise
adding chloroacetyl chloride in a molar ratio of chloroacetyl
chloride to methoxy polyethylene glycol MPEG2000 of 10:1 to 1:1,
stirring in the dark overnight, precipitating in diethyl ether to
obtain a product, dissolving the product in water, adjusting the pH
value to 6, extracting 3 times with 20 ml of dichloromethane, and
adding diethyl ether and washing to obtain a precipitate B;
[0009] and (3) using dichloromethane as a solvent, adding the white
crystal A and the precipitate B in a molar ratio of 0.2:1 to 1:0.2,
and adding KI in a molar ratio of KI to white crystal A of 0.1:1 to
1:0.1, and adding potassium carbonate of 0.002 mol/L to 0.2 mol/L,
reacting for 2-48 h, filtering, and adding the filtrate to diethyl
ether to precipitate a precipitate C, and the precipitate C is the
water-soluble ferric ion fluorescent probe.
[0010] A schematic representation of the reactions involved in the
above preparation steps is as follows:
##STR00003##
[0011] The beneficial effects of the present invention:
[0012] 1. The water-soluble ferric ion fluorescent probe of the
present invention has good water solubility and does not require
the addition of an organic solvent when used, which makes it
possible to directly measure ferric ion in an aqueous environment
containing living organisms.
[0013] 2. The fluorescent ion probe of the present invention has
excellent selectivity to ferric ion, and has good anti-interference
ability to common metal ions such as Fe.sup.3+, Fe.sup.2+,
Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Ca.sup.2+, Cd.sup.2+, Mg.sup.2+,
Pb.sup.2+, Hg.sup.2+, K.sup.+ and Na.sup.+, etc.
[0014] 3. The detection of ferric ion by the fluorescent ion probe
of the present invention can be observed by the naked eye, which
does not require complicated instruments.
[0015] 4. The fluorescent ion probe of the present invention can
penetrate the cell membrane and bind to Fe.sup.3+ in the cell, and
can be used for monitoring Fe.sup.3+ in the internal environment of
living cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a fluorescence emission spectrum (excitation
wavelength was 500 nm) of a solution after adding different metal
ions to an aqueous solution of the water-soluble ferric ion
fluorescent probe of the present invention.
[0017] FIG. 2 is a photograph of two sample bottles under different
light (left under sunlight, right under an ultraviolet lamp) (in
both the bottles were aqueous solutions of the water-soluble ferric
ion fluorescent probe of the present invention, the left bottle was
not added with Fe.sup.3+, and the right bottle was added with
Fe.sup.3+).
[0018] FIG. 3 is a confocal fluorescence photograph of HeLa cells
after incubation for 5 h in medium containing different samples.
Wherein, the first row of photographs are cells cultured only with
the water-soluble ferric ion fluorescent probe of the present
invention, and the second row of photographs are cells sequentially
cultured with Fe.sup.3+ and the water-soluble ferric ion
fluorescent probe of the present invention. The first column is a
fluorescent photograph, the second column is a bright field photo,
and the third column is a superimposed photo of the first two
columns.
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLE 1
[0019] The preparation method of ferric ion fluorescent probe
compound C comprises the following steps:
[0020] Synthesis of rhodamine 6G derivative (A): 2 mmol of
rhodamine 6G was dissolved in 20 ml of hot ethanol, and
1,3-propanediamine was dropwise added, and the mixture was heated
under reflux for 6 h. The solution was cooled to room temperature,
and a solid was precipitated and recrystallized with acetonitrile
to give a white crystal A. (.sup.1H-NMR (CDCl.sub.3): .delta. 7.95
(d, 1H), 7.47 (t, 2H), 7.05 (d, 1H), 6.34 (s, 2H), 6.23 (s, 2H),
3.50 (t, 2H), 3.24 (t, 4H), 2.39 (t, 2H), 1.90 (s, 6H), 1.36 (t,
6H), 1.19 (m, 2H))
[0021] Synthesis of methoxy polyethylene glycol chloroacetate (B):
10 g (5 mmol) of methoxy polyethylene glycol (molecular weight
2,000) was dissolved in 20 ml of anhydrous dichloromethane, and
2.09 ml (15 mmol) of triethylamine, 7.95 ml (100 mmol) of
chloroacetyl chloride was dropwise added under a N2 atmosphere, and
the mixture was stirred overnight in the dark. A product was
precipitated from diethyl ether. The product was dissolved in
water, adjusted to pH value 6, extracted 3 times with 20 ml of
dichloromethane, and added with diethyl ether to precipitate a
precipitate B. (.sup.1H-NMR (CDCl.sub.3) (4.20 (COOCH2), 4.15
(CH2Cl), 3.3-3.7 (OCH2CH2O))
[0022] Synthesis of fluorescent probe (C): 10 mg (0.02 mmol) of
rhodamine 6G derivative, 4.4 mg (0.026 mmol) of KI, 15 mg (0.11
mmol) of potassium carbonate, 10 mg (0.005 mmol) of PEG, and 5 ml
of dichloromethane, were reacted for 10 h under N2, and filtered,
and the filtrate was added to diethyl ether to precipitate a
precipitate C. (.sup.1H-NMR (CDCl.sub.3) (.delta. 7.95 (COCCH),
7.47 (CHCHCH), 7.05 (CHCHC), 6.34 (CCHCC), 6.23 (CCHCO), 4.20
(COOCH2). 4.15 (CH2Cl), 3.3-3.7 (OCH2CH2O, NCH2, OCH3), 3.24
(CH2CH3), 2.39 (NHCH2), 1.90 (CCH3), 1.36 (CH2CH3),
1.19(NCH2CH2))
EXAMPLE 2
Performance Experiment of Ferric Ion Fluorescent Probe Compound
[0023] 1) Selectivity of the Ferric Ion Probe
[0024] To the parallel groups of water with a pH value of 7.0, the
above precipitate C was added, respectively, Fe.sup.3+, Fe.sup.2+,
Cu.sup.2+, Zn.sup.2+, Ni.sup.2+, Ca.sup.2+, Cd.sup.2+, Mg.sup.2+,
Pb.sup.2+, Hg.sup.2+, K.sup.+, and Na.sup.+ were added to each
group in turn, 5 .mu.M each, and the last group was not added as a
control group. The fluorescence emission spectra of each group
(excitation wavelength was 500 nm) were measured. It was found that
the fluorescence intensity was significantly enhanced after the
addition of Fe.sup.3+ compared with the control without ions; and
the fluorescence intensity of the solution was substantially
unchanged after the addition of other ions (as shown in FIG. 1).
The results show that the precipitate C has excellent selectivity
for Fe.sup.3+.
[0025] 2) Detection of Ferric Ion by the Ferric Ion Fluorescent
Probe
[0026] As shown in the left figure of FIG. 2, under the daylight, a
water sample of the precipitate C was added to the sample bottle on
the left, and the water sample in the sample bottle on the right
was added with the precipitate C and Fe.sup.3+. The two bottles
were illuminated with an ultraviolet lamp, and the photograph in
the right figure was obtained. It can be seen that the precipitate
C was colorless and clear transparent in water, which proves that
its water solubility is better than that of the general
rhodamine-based fluorescent probe. On the other hand, the
precipitate C changed from colorless to flesh pink when it
encountered Fe.sup.3+ in water, and the color change was very
obvious and visible to the naked eye. It glowed bright yellow
fluorescence under the illumination of an ultraviolet lamp. It can
be seen that the detection of Fe.sup.3+ by the precipitate C can be
judged by the naked eye without using an instrument.
[0027] 3) Cellular Experiment of the Ferric Ion Probe
[0028] HeLa cells were cultured in medium DMEM for 5 h in a
37.degree. C. incubator, one group was added with Fe.sup.3+ (10
.mu.M) in the medium, and another group was not added with
Fe.sup.3+ as a control group. Then, Fe.sup.3+ which did not enter
the cells was washed away with a phosphate buffer solution having a
pH value of 7.4, respectively. The two groups of cells were
separately transferred to DMEM supplemented with C (10 .mu.M) and
further cultured. After 30 minutes, the cells were rinsed with a
phosphate buffer solution and then observed with a confocal
fluorescence microscopy. Fluorescence imaging of the two groups is
shown in FIG. 3. It was found that almost no fluorescence was
observed in the control group (the first row of photographs in FIG.
3), i.e., the cells which were not previously cultured with
Fe.sup.+, and the cells cultured in the medium containing Fe.sup.3+
(see the second row of photographs in FIG. 3) showed fluorescence.
The results show that the ferric ion fluorescent probe of the
present invention is capable of penetrating the HeLa cell membrane
to bind to Fe.sup.3+ in the cell. This ability to penetrate cell
membranes and the ability to enhance fluorescence due to binding to
Fe.sup.3+ may play an important role in monitoring the
concentration of Fe.sup.3+ in cells to study toxicity or biological
activity in living cells.
* * * * *